Time-of-flight mass spectrometers rapidly acquire a sequence of individual time-of-flight spectra. However, to avoid saturation effects for the most intense ion signals within the spectrum, the spectra must only contain a few hundred ions at most, and therefore they have a lot of empty mass ranges and are highly scattered. For substances whose concentration is low, an ion is only measured in every tenth, hundredth or even thousandth individual time-of-flight spectrum. Hundreds or even thousands of these individual time-of-flight spectra, which at present can be acquired at frequencies of up to two thousand spectra per second, are then immediately processed to create a sum spectrum in order to obtain usable time-of-flight spectra with a large dynamic measurement range for the ion species of the various analyte substances. When the term “mass spectrum” is used in this document, this designates this sum spectrum. Nowadays between 50 and 5000 individual spectra, depending on the dynamic range of measurement required, are regularly added together to form a mass spectrum.
The term “ion signal” here refers to the part of a mass spectrum that contains “identical ions”. This calls for a closer definition of the term “identical ions”. In a mass spectrometer with ultra-high resolution, an ion signal only contains ions comprised of the same isotopes; ions with a different isotopic composition, even if they have the same nominal mass, can generally be separately measured, and form a different ion signal. In a mass spectrometer that “only” offers high resolution, an ion signal may contain all the ions with the same nominal mass; those ions whose isotopic composition results in different nominal masses form separate ion signals, even in the mass range of some thousands of daltons. In lower-resolution mass spectrometers, an ion signal, particularly in the high-mass range, can encompass all the ions with the same constituent elements i.e. all the ions that have the same molecular formula. The envelope of the isotopic distribution is then measured as the ion signal. An ion signal is also referred to as an “ion peak”.
In particular, time-of-flight mass spectrometers operated in linear mode, which measure organic ions with masses in the range of some thousands of daltons, can no longer resolve the isotopic signals of different nominal masses. The ion signals therefore here comprise all the ions in the isotopic distribution, i.e. all the ions with the same molecular formula; the ion signal has essentially the shape of the envelope of the isotopic distribution. FIGS. 1, 2 and 3 illustrate three such distributions of the isotopic signals over the nominal masses for singly charged protein ions with masses of 5000, 12,000 and 20,000 daltons. On the other hand, good time-of-flight mass spectrometers operating in reflector mode achieve mass resolutions of R=m/Δm=40,000; generally speaking they can then resolve ion signals of different nominal masses, and the isotope distributions of FIGS. 1 to 3 therefore appear resolved according to nominal masses.
In order to measure the time-of-flight mass spectra, the electrical currents created at the ion detector by the ions after they have passed through the flight path are first amplified by secondary electron multipliers by factors between 105 and 107, and are then sampled using special digitization units known as “transient recorders”. These units contain very fast analog-to-digital converters (ADC); nowadays they operate with sampling rates of around 4 gigasamples per second (GS/s), and higher sampling rates of up to around 10 gigasamples per second are at present under development. The limit to the sampling rate results at present not from the ADC itself, but from the further processing of the measured values to generate an averaged or summed mass spectrum. Usually each measurement is only digitized to a depth of eight bits, therefore only encompassing values between 0 and 255; a good dynamic range of measurement covering five or six orders of magnitude can therefore only be achieved by summing hundreds or thousands of individual spectra into a mass spectrum. Even if it became possible in the future to raise the digitization depth of the ADC to ten or even twelve bits, it would still be necessary to add together many hundreds of or thousands individual spectra in order to obtain a mass spectrum with a sufficiently high dynamic measuring range of four to six orders of magnitude.
For this acquisition technique it is obvious that the ion detector must, on the one hand, register every single ion, but on the other hand must also deliver the highest possible dynamic range of measurement in each individual spectrum. Furthermore, in the individual time-of-flight spectra, the analog-to-digital converter must not become saturated for any of the ion signals, and the ion current must therefore be limited. In order not to lose any ions, but at the same time to achieve a wide measuring range, the amplification provided by the secondary electron multiplier must be adjusted very accurately. Methods for optimum adjustment of the amplification of the secondary electron multiplier are disclosed in U.S. Published Application 20090206247. Because of the Poisson distribution of the secondary electrons generated by the impact of an ion, it is favorable for a single ion to yield a signal that generates a mean measured value of at least about 2 or 3 counts in the ADC. This, however, restricts the intensity dynamic range within an individual time-of-flight spectrum to about two orders of magnitude: from around 2.5 counts up to 255 counts. Only a few percent of individual ions, however, are lost with this adjustment.
This optimum adjustment of the secondary electron multiplier, however, only applies to ions of a selected mass-to-charge ratio m/z, since the sensitivity of the secondary electron multiplier depends on the mass, and declines in approximate proportion to 1/√(m/z). If the amplification of the secondary electron multiplier is adjusted, for instance, in such a way that the 2 or 3 counts mentioned above are achieved for a singly charged ion with a mass m=20,000 daltons, in order that no ions of high mass are lost, then a singly charged ion with a mass m=2000 daltons will already yield 8 counts, and the measuring range for ions of this mass is restricted to merely one and a half orders of magnitude between 8 and 255 counts.
Restricting the ion current in order to avoid saturation effects is, however, not always without difficulty; the restriction can itself also have very unfavorable effects. In the case, for instance, of the ionization of analyte molecules by matrix-assisted laser desorption under consideration here, not all the analyte molecules are ionized to the same degree if the laser energy is reduced for the sake of a reduced ion current. Analyte molecules with low proton affinity are not ionized unless the energy density of the laser light pulse in the laser spot on the sample preparation is high enough. Then, however, many ion signals may already be saturated. These problems occur in particular when analyzing protein mixtures. If, for analytic reasons, it is required to ensure that all the proteins in the mixture have about the same probability of being ionized, it is necessary to generate a high energy density within the laser spot. With today's acquisition techniques it is not usually possible to achieve this, since it would then cause many ion signals to reach saturation.
At the place where the laser light is focused on the sample, the “laser spot”, a plasma consisting of heated, vaporized matrix substance is formed by every laser shot. Each sample consists of tiny crystals of the matrix substance, in which the protein molecules are embedded at very low concentrations on the scale of a hundredth of a percent or lower. The plasma very quickly reaches a maximum temperature, and then cools down again very quickly through adiabatic expansion into the surrounding vacuum. As in any hot plasma, some of the molecules of the matrix substance are ionized in this plasma. The matrix substances are selected in such a way that their ions easily donate protons to the much larger protein molecules that have a higher proton affinity. If, as a result of a lower energy density in the laser spot, the plasma generated is only moderately hot, not enough proton donors are present to ionize all the protein molecules; a competitive situation develops, in which some proteins are favored due to their higher proton affinity while others are disadvantaged. It is recognized that there are real “killer substances” for MALDI, whose inclusion can suppress the ion signals from other substances.
The number of ions in the plasma depends heavily on the maximum temperature reached. Investigations can be found in the literature that show that the number of analyte ions in the plasma rises, over a wide range of energies, with the sixth or even the seventh power of the energy density in the laser spot. Practical experience in the use of MALDI mass spectrometers confirms this observation: the ion current from the analyte molecules rises by six or seven percent when the energy density in the laser spot is raised by one percent. However, if the energy density is simply increased while using the usual sizes of laser spot, so many ions are created by each laser light shot that the ion detector goes into saturation for many of the ion signals in an individual spectrum. As a result, a good (sum) mass spectrum cannot be generated, in addition to which many of the signals that have gone into saturation become so wide that it is no longer possible to tell whether they represent one signal from a single ion species, or whether perhaps they are hiding the signals from two, three or four ion species.
MALDI therefore entails a dilemma between, on the one hand, avoiding saturated signals and, on the other hand, ionizing all the substances in a mixture equally.
Modern laser systems such as the “Smart Beam” laser from Bruker Daltonik GmbH, Bremen, may offer an improvement here even if they cannot provide a complete remedy. At every shot, these laser systems can generate either one or more laser spots with a very small diameter; and because of the small area, only a limited number of ions are delivered, even at high energy density in the laser spot. The duration of the laser light pulses is also optimized to provide the highest ion yield. Laser light pulses from these lasers vaporize extremely little material, but the ionization yield is high, and there is a high probability that molecules with low proton affinity become ionized. But even here, under optimum ionization conditions for the protein molecules, a large number of signals still become saturated.
The acquisition technique used until now is illustrated here with the example of a method used for the identification of microorganisms. The microorganisms, or “microbes” for short, are identified through an analysis of their soluble cell components (primarily proteins) in time-of-flight mass spectrometers operated in linear mode and with the ionization by matrix assisted laser desorption mentioned above. Because it is important for this method to reliably detect the proteins that are present on the basis of their ions, even when they are in mixtures with the proteins from other microbes, the dilemma described above is particularly acute here. For this reason, the method is now described in somewhat more detail:
The generation of the mass spectra of the microbial components usually starts from a cleanly separated colony on a solid, typically gelatinous culture medium or from a centrifugal sediment (pellet) extracted from a liquid nutrient medium. Using a small object such as a wooden toothpick, a tiny quantity of microbes is picked up from the selected colony or from the sediment, and placed on the mass spectroscopic sample support. This sample is then sprinkled with an acidified solution of a conventional matrix substance, the purpose of the matrix substance being to assist the later ionization of the microbial components. The acid in the matrix solution now attacks the cell walls and weakens them; the organic solvent penetrates into the microbial cells causing them to burst due to osmotic pressure, so releasing the soluble proteins. The sample is then dried by evaporating the solvent, leading to crystallization of the dissolved matrix material. In this process, soluble protein molecules are embedded, separately from one another, within the matrix crystals. A number of different types of crystalline organic acids, such as HCCA (α-cyano-4-hydroxycinnamic acid), can be used as the matrix substance.
Instead of transferring whole microbes with the toothpick, microbes that have been cleaned by washing and centrifuging can also be decomposed in the centrifuge tube; strong acids that break down even hard microbial cell walls can be used here. Centrifuging precipitates out the insoluble components such as cell walls, so that these can no longer interfere with the mass spectrometric analysis. About one microliter of the supernatant decomposition fluid is now applied to the mass spectroscopic sample support, where it is dried. By applying a further coat of a suitable matrix solution and drying once again, the analytic sample is made ready on the sample support, and the protein molecules are embedded in the matrix crystals. These sample preparations with external decomposition give mass spectra that are practically the same as those obtained from the usual preparation process on sample supports. The mass spectra obtained from these decomposition processes are, however, cleaner than the usual preparations performed on the sample supports; they exhibit less interfering background, and are therefore more suitable for detecting the target microbes, even in mixtures with other microbes.
The sample preparations that have been dried on the sample supports, i.e., the matrix crystals with the embedded protein molecules, are then subjected to pulsed UV laser light in the ion source of the mass spectrometer; each pulse of laser light gives rise predominantly to singly charged ions of the protein molecules, which can then be measured in the mass spectrometer according to ion mass. Preferably, specially developed, highly sensitive MALDI time-of-flight mass spectrometers with a very simple design without reflectors are used for this purpose. Ions in the range of masses between 2000 and 20,000 daltons are measured.
Due to the particularly high detection sensitivity, the mass spectra of the microbe proteins are acquired by these time-of-flight mass spectrometers operating in linear mode. In other words, no energy-focusing reflector is used, although the mass resolution and mass trueness of the spectra from time-of-flight mass spectrometers operating in reflector mode is significantly better. As a result, the ion signals correspond, in the stated mass range, approximately to the envelopes of the isotope distributions, as illustrated in FIGS. 1 to 3.
The mass spectrum of a microbial isolate is the frequency profile of the mass values of the protonated molecular ions of the soluble cell components of the microbes. This nearly always involves mixtures of protein ions. Each pulse of laser light generates one single mass spectrum, which is measured in the time-of-flight mass spectrometer in less than 100 microseconds; but in the prior art, this spectrum must only contain signals with no more than a hundred ions per measuring cycle, i.e., every 0.25 nanoseconds at a sampling rate of 4 gigasamples/second. In order to obtain mass spectra with lower noise and a higher measuring range, it is usual to add together some hundreds or a few thousand of these individual mass spectra to form a sum spectrum. It is always this sum spectrum that is referred to by the term “mass spectrum of a microbe” or, more simply, “microbe spectrum”. Thanks to the high rate of laser bombardment (at present, up to two kilohertz), it only takes a few seconds to acquire such a microbe spectrum. A sample support plate having 48, 96 or even 384 prepared samples can be measured automatically in less than half an hour.
The protein profile represented by each of these microbial spectra is highly characteristic of the particular microbe type because every species of microbe produces its own, genetically programmed proteins, each with characteristic masses. The frequency of the individual proteins in the microbes, inasmuch as they can be measured by mass spectroscopy, are also largely genetically programmed through the control of their production by other proteins, and only depend to a minor extent on the nutrient medium or on the maturity of the colony, provided they are not forming spores. The protein profiles are characteristic for microbes in rather the same way as fingerprints are for people. As a result it is possible to identify the microbes through a similarity analysis against reference spectra held in a reference library.
The spectra are evaluated using programs supplied by the manufacturers of the mass spectrometers. These programs are based on similarity analyses of a measured microbe spectrum against reference mass spectra from specially validated spectral libraries. A similarity coefficient is calculated for each reference spectrum. If the highest similarity coefficient found exceeds a specified similarity threshold, this constitutes unambiguous detection of the microbial species to which the corresponding reference spectrum belongs. Special similarity thresholds are used for the identification of family, genus or species.
Acquisition in accordance with the prior art yields neither a high concentration accuracy nor good reproducibility of the ion signals in the mass spectra. Low-level contamination of the sample preparations with salts, with substances having a high proton affinity, or with small quantities of other microbial species can sharply modify the mass spectra. For this reason, the prior art only assigns a very subsidiary role to the intensity of the ion signals in the calculation of the similarity coefficients. An improvement in the concentration accuracy and in the reproducibility of the mass spectra would significantly improve the method for identifying the microbes.
It should be stressed here that the mass spectrometric method has until now been applied only to the identification of microbes of unknown kind in samples. Usually the microbe isolates used for the preparation of these samples are obtained from well-separated colonies grown an agar plates. The identification of two, or at most three, microbial species in a mixture of these two or three microbial species has also been disclosed German Patent Application DE 10 2009 007 266 A1. It is, however, an as-yet unused strength of the mass spectrometric method that, under certain conditions, it can unambiguously and reliably detect the presence or absence of a specific microbial target species in rather more complex mixtures of five, ten or more microbial species. An improvement in the reproducibility and the concentration accuracy is, however, indispensable for this kind of rapid detection of specified microbes in foodstuffs, bathing water, stool or other samples.
Although the problem of mixture analysis has been described on the basis of the analysis of microbe proteins in linear time-of-flight mass spectrometers, it is not restricted to this. The problem also occurs in high-resolution MALDI time-of-flight mass spectrometers operated with reflectors for the analysis of proteins. Quite generally, therefore, a solution that will improve the analysis of mixtures is desirable for MALDI time-of-flight mass spectrometers.
An objective of the invention is to solve the MALDI dilemma, described above, between signal saturation and concentration accuracy, and to improve the reproducibility and also, if possible, the dynamic measuring range of the spectral acquisition in MALDI time-of-flight mass spectrometers.